Molecular Analysis of Multiple otiTs in the Gonococcal Mobilizable R-Plasmids

细菌病原体之间的质粒动员的重要性正变得越来越重要 显然，像淋球菌这样的多重耐药细菌正在以惊人的速度出现。在淋球菌中发现了3个大小分别为7.4、5.6和5.2kb的R-质粒。7.4和5.6kb的R-质粒含有一个1.9kb的动员盒，带有MOBA和mobC，以及一个168bp的基因间隔区，带有一个功能性ORIT。5.2kb的R-质粒没有这个盒，但可以被41kb的TetM和R6K接合质粒动员。因此，我们的假设是，淋球菌R-质粒的混杂是由于存在两个或两个以上的转移来源，与其相关的Mob蛋白的分子相互作用引发了不同家族的接合质粒的动员。我们建议通过序列缺失和定点突变来鉴定基因间隔区的功能ORIT。利用共居重组质粒提供的MOBA蛋白在体内的互补，将开发出一种强大的动员选择实验。对MOBA和MOBC基因启动子序列的遗传分析将确定相邻的MOB蛋白在动员转移过程中的转录调控作用。当在接合过程中被动员时，5.2kb的R-质粒在1.9kb的盒之外使用不同的ORIT。第二个功能ORIT和与动员5.2kb R-质粒相关的反式作用蛋白将通过使用R6K和41kb TetM结合质粒的互补分析来鉴定。这些结合质粒将提供识别第二转移来源的外源Mob蛋白。新的独特ORIT和相关MOB蛋白的生物学特性将通过测序、功能表征和使用基因库数据库的比较分析来确定。这项研究的结果将有助于开发创新的策略，以控制由于获得R-质粒而导致的抗生素水平耐药基因传播，以及淋球菌和其他病原菌出现多重耐药。

The importance of plasmid mobilization among bacterial pathogens is becoming increasingly apparent as multidrug resisistant bacteria, like Neisseria gonorrhoeae, are emerging at an alarming rate. Three R-plasmids of 7.4, 5.6 and 5.2 Kb have been identified in gonococcus. The 7.4 and 5.6 Kb R-plasmids contain a 1.9 Kb mobilization cassette with mobA, mobC and a 168 bp intergenic region with a functional oriT. The 5.2 Kb R-plasmid lacks this cassette but can be mobilized by the 41 Kb TetM and R6K conjugative plasmids. Therefore, our hypothesis is that the promiscuity of the gonococcal R-plasmids is due to the existence of two or more origins of transfer, and the molecular interactions with its associated Mob proteins trigger plasmid mobilization by different families of conjugative plasmids. We propose to identify the functional oriT in the intergenic region by sequence deletion and site-directed mutagenesis. A powerful selection assay for mobilization will be developed using in vivo complementation with MobA protein provided from a co-resident recombinant plasmid. Genetic analysis of the promoter sequence for mobA and mobC genes will establish the role of transcriptional regulation by adjacent Mob proteins during the mobilization transfer. The 5.2 Kb R-plasmid uses a different oriT outside from the 1.9 Kb cassette, when it is mobilized during conjugation. This second functional oriT and trans-acting proteins associated with the mobilization of the 5.2 Kb R-plasmids will be identified by complementation assays using the R6K and the 41 Kb TetM conjugative plasmids. These conjugative plasmids will provide the exogenous Mob proteins that recognize the second origin of transfer. The biological properties of the new distinct oriT and related Mob proteins will be determined by sequencing, function characterization and comparative analysis using Gene Bank databases. The result of this research will contribute to the development of innovative strategies for control of horizontal antibiotic resistance gene transmission due to R-plasmid acquisition and the emergence of multiresistance in N. gonorrhoeae and other pathogenic bacteria.